It is found that m-FPEABr allows the transformation of low-n phases to high-n phases during the annealing process, leading to the suppression of n = 1 phase and increasing higher-n phases with improved crystallinity. The rational phase distribution results in the formation of multiple quantum wells (MQWs) in the m-FPEABr based films. The carrier dynamics study reveals that the resultant MQWs enable rapid energy funneling from low-n phases to emission centers. As a result, the green PeLEDs achieve a peak external quantum efficiency of 16.66% at the luminance of 1279 cd m−2. Our study demonstrates that the fluorinated organic cations would provide a facile and effective approach to developing high-performance PeLEDs.Metal halide perovskites have become a research highlight in the optoelectronic field due to their excellent properties. The perovskite light-emitting diodes (PeLEDs) have achieved great improvement in performance in recent years, and the construction of quasi-2D perovskites by incorporating large-size organic cations is an effective strategy for fabricating efficient PeLEDs. Here, we incorporate the fluorine meta-substituted phenethylammonium bromide ( Metal halide perovskites have attracted great attention in the field of luminescent materials and devices because of their excellent characteristics, such as their high photoluminescence quantum yields (PLQYs), tunable optical band gap, narrower emission spectra, and solution processability ,4,5,6,7.3 possess low exciton binding energy, which is not conducive to radiative recombination and will result in low luminous efficiency  (PTAA)  from Xi’an Polymer Light Technology Corp. . We purchased PbBr2 (99.999%), CsBr (99.999%), poly(9-vinylcarbazole) (PVK) , DMSO , ethanol (anhydrous) and chlorobenzene  from Sigma-Aldrich . Nickel acetate tetrahydrate (Ni(CH3COO)2·4H2O) (99.9%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. . Ethanolamine (99%) was purchased from ACROS, , and 1,3,5-tris(2-N-phenylbenzimidazolyl)-benzene) (TPBI) (≥99%) and LiF (≥99%) were purchased from Jilin OLED Photoelectric Material Corporation . The molecular structures of m-FPEABr and PEABr are shown in We purchased 3-fluorophenylethylammonium bromide  filter.The precursor solution of NiOx was prepared according to the previous method ,37. Nickm-FPEABr, PEABr, CsBr, and PbBr2 in DMSO at the desired molar ratio in a N2-filled glove box. And the concentrations of CsBr and PbBr2 were set at 0.3 M. The solutions were stirred at 45 °C overnight, and then was left to stand for 4 h at room temperature and filtered with 0.22 µm PTFE filters before use.The precursor solutions of perovskites were prepared by dissolving x precursor solution was spin coated on an ITO anode at 4000 r.p.m. and annealed at 350 °C for 60 min in air. The PTAA (8 mg mL−1 in chlorobenzene) was spin coated at 4000 r.p.m. and dried at 170 °C for 30 min in a N2-filled glove box. Pure chlorobenzene was then spin coated to obtain an ultrathin PTAA layer. After that, the PVK (8 mg mL−1 in chlorobenzene) was spin coated on PTAA and then also dried at 170 °C for 30 min. Perovskite films were spin coated at 4000 r.p.m for 120 s and annealed at the desired temperature for 10 min. Finally, the samples were transferred into a thermal evaporator, and the electron-transport layer of TPBi (40 nm), the electron-injection layer of LiF (1 nm), and the cathode of Al (100 nm) were deposited consecutively onto the perovskite film under a high vacuum of less than 4 × 10−4 Pa. The active area of the devices is 8 mm2, which is determined by the overlap area of Al cathode and ITO anode.The schematic device structure of the PeLEDs is shown in The ultraviolet-visible absorption spectra were measured with a HP 8453 spectrophotometer. The photoluminescence (PL) spectra were measured with a Shimadzu RF-5301PC spectrometer with an excitation wavelength of 365 nm. The PLQYs were measured by a commercialized measurement system XPQY-EQE-350-1100, from Guangzhou Xi Pu Optoelectronics Technology Co., Ltd. with a 365-nm LED as the excitation light source. The time-resolved photoluminescence (TRPL) decay curves of the perovskite films were measured by Edinburgh FLS980 with a 375-nm laser. XRD measurements were performed with a RIGAKU Smart lab (9 kW) X-ray diffractometer with Bragg–-Brentano focusing, a diffracted beam monochromator and a conventional Cu target X-ray tube (λ = 1.5405 Å) set to 40 keV and 30 mA. The grazing incidence wide-angle X-ray scattering (GIWAXS) measurement (incidence angle = 0.3°) was performed on the BL14B1 beamline of the Synchrotron Radiation Facility (SSRF) in Shanghai, China.2-filled chamber.The broadband femtosecond transient absorption (TA) spectra of the perovskite films were taken by using the Ultrafast System HARPIA TA spectrometer . A Yb: KGW amplifier  supplied laser beams centered at 1030 nm with pulse duration of ~100 fs, pulse repetition rate of 40 kHz, and a maximum pulse energy of 100 µJ. The output of the amplifier was split into two streams of pulses. One was used to drive an optical parametric amplifier  to obtain the pump beam. The residual stream was directed into an ultrafast spectroscopic system  to generate the white light continuum probe beam. In the spectrometer, the pump chopped at the frequency of 20 kHz was spatially and temporally overlapped with the probe beam on the sample. The pump beam was focused onto the sample with a beam size of 300 µm, which overlapped with the smaller-diameter (200 µm) probe beam. The pump and probe pulses were crossed on the sample's surface, and the transmitted probe beam was recorded by using a CCD linear Si detector coupled to a monochromator. The perovskite samples were placed in a NThe electroluminescence (EL) characteristics of current density-luminance-voltage (J-L-V) were performed simultaneously by using a computer-controlled source meter (Keithley 2400) equipped with a luminance meter . The EL spectra were recorded with a spectrometer . The EQEs were calculated from the luminance, current density, and EL spectra, assuming a Lambertian distribution. All results from the devices were measured in the forward-viewing direction without any out-coupling enhancement techniques. All EL measurements were carried out at room temperature under ambient conditions.m-FPEABr content on the device performance is first studied. We employ the perovskite precursors of m-FPEABr: CsBr: PbBr2 with the stoichiometric ratio of x: 1: 1  to construct the quasi-2D perovskite films as the emissive layers (EMLs). The perovskite EMLs are prepared by the facile one-step spin-coating method without any anti-solvent treatment, and the annealing temperature is kept at 110 °C. The EL characteristics of the PeLEDs with various m-FPEABr contents are shown in m-FPEABr and PEABr contents are summarized in m-FPEABr or PEABr shows the extremely poor EL performance, achieving only a maximum EQE of 0.05% and a maximum luminance of 72 cd m−2. The incorporation of large cations can lead to a great improvement in the EL performance and a slight blue shift in the emission peak. The luminance of PEABr-based PeLEDs is increased to 7535 cd m−2 at the PEABr content of 40%, but it keeps decreasing with the rise of PEABr content. Moreover, the maximum EQEs of the PEABr-based devices are all below 3%, still showing poor EL performances. As for the PeLEDs based on m-FPEABr, the device efficiency is greatly improved from 1.38% to 8.53% as the m-FPEABr content is increased from 40% to 60%. When the m-FPEABr content reaches 80%, the quasi-2D PeLED achieves a maximum EQE of 11.41% and a maximum luminance of 10,603 cd m−2 with the EL peak at 514 nm. Further increasing the m-FPEABr content obviously results in the drastic decrease in both EQE and luminance. The above results demonstrate that the incorporation of both PEABr and m-FPEABr can improve the device performance, but the maximum luminance will significantly decrease if adding too many organic cations. The decreased luminance would originate from the reduced conductivity of the perovskite films with too much insulating organic cations, which is in agreement with the reduced current density as shown in m-FPEABr show the best EL performance; we therefore set the organic cation content as 80% in the precursor solutions to construct quasi-2D perovskite for the subsequent study.The large organic cations have a great influence on the structural and optoelectronic properties of quasi-2D perovskites, which can be used to manipulate the performance of quasi-2D PeLEDs. Here, the effect of m-FPEABr EMLs annealed at various temperatures are given in m-FPEABr and PEABr show a significant upward trend in the maximum luminance; their maximum EQEs increase first and then decrease. It is noteworthy that the champion device based on m-FPEABr achieves a high EQE of 16.66% at the annealing temperature of 120 °C, and its maximum luminance is obtained as 10,532 cd m−2. When the annealing temperature is increased up to 140 °C, the device based on m-FPEABr can realize the maximum luminance of exceeding 28,000 cd m−2, even though its maximum EQE is dramatically reduced to 6.55%. As for the PEABr-based devices, a decent EL performance with the maximum EQE of 6.25% can be observed at the annealing temperature of 120 °C, which is still far inferior to that of the champion device based on m-FPEABr. These results clearly indicate that the fluorine meta-substitution of PEABr enables the quasi-2D perovskites to significantly improve the EL performance. We believe that the device performance can be further enhanced by optimizing the device structure, such as improving the electrical properties of the transport layers by doping 4− octahedra and large organic cation, which allows us to focus on the interaction between inorganic octahedra and organic cations [fH0K is determined to be −0.76 eV per unit for m-FPEA2PbBr4 and −0.48 eV per unit for PEA2PbBr4. This suggests that m-FPEABr is much easier than PEABr to form the 2D structure, which is consistent with the absorption results of the quasi-2D perovskites before annealing (m-FPEABr perovskite during the spin coating. After annealing treatment, the low-n phases in the m-FPEABr perovskite are gradually decreased, especially for the n = 1 phase, which can be attributed to the transformation of the low-n phases to the high-n phases. From the above discussion, we can clearly see that the phase distribution has a significant effect on the carrier dynamics of the quasi-2D perovskites. Remarkably, the difference in the phase distribution originates from the introduction of a single fluorine atom on the benzene ring of phenylethylamine cation. This molecular structure perturbation should affect the intermolecular interactions. For the purpose of studying the effect of the molecular structure of organic cations on the assembly of perovskite structure, we estimated the formation enthalpy (Δ cations . The ΔHfnnealing . Accordim-FPEABr and CsPbBr3 was demonstrated to form the quasi-2D perovskites with homogeneous phase distribution, which enable efficient energy funneling from low-n phases to high-n phases in the MQWs. By contrast, the control quasi-2D perovskites based on the widely used PEABr presented inhomogeneous phase distribution with significantly fewer low-n phases. More interestingly, the high-n phases in the m-FPEABr-based quasi-2D perovskites displayed more ordered crystal orientation preferentially among the out-of-plane direction. Additionally, the grain sizes became larger when using m-FPEABr to replace PEABr in the quasi-2D perovskite films, which can lower the effect of defects on the energy funneling and carrier transporting. By manipulating the phase distribution and crystallinity of the quasi-2D perovskites by m-FPEABr, the green PeLEDs achieved a peak EQE of 16.66%, which is among the best EL performances of the perovskites without anti-solvent treatment or addition of a passivation agent. Our study should shed light on the rational regulation of crystallization kinetics to realize efficient energy funneling in quasi-2D perovskite films for high-performance PeLEDs.We employed a fluorinated organic cation to prepare quasi-2D perovskite films with an MQW structure by simple one-step spin coating without anti-solvent treatment for efficient green PeLEDs. The combination of


Despite several limitations, imaging is still the gold standard in evaluating the treatment effect in metastatic colorectal cancer, and the best response is often achieved several months after treatment start. The present study investigated methylated circulating tumor DNA (ctDNA) in the blood and found that a reduction in ctDNA after one treatment cycle was a better marker of progression-free survival than imaging. Furthermore, ctDNA increases in the blood before progression are visible on imaging, allowing for a change in treatment at an earlier time point. Methylated ctDNA is an early indicator of treatment benefit and a promising endpoint in clinical trials.neuropeptide Y circulating tumor DNA (meth-NPY) as an early biomarker for treatment effect and monitoring in 70 mCRC patients receiving first-line treatment in the FOLFOXIRI-Toco trial. Meth-NPY was analyzed using droplet digital PCR, and the response rate was defined as the fraction of patients converting from a baseline detectable level to an undetectable level after the first treatment cycle (responders). A significant increase in meth-NPY was defined as a value with no overlap between the 95% CI of the current and preceding measurement. Progression-free survival (PFS) was significantly longer in meth-NPY responders compared to non-responders, 10.1 and 7.6 months, respectively . Patients with response according to RECIST 1.1 had a PFS of 10.1 compared to 7.3 months for non-responders . A significant increase in meth-NPY was found with a median of 49 days before radiological progression. In conclusion, early meth-NPY response proved superior to response according to RECIST 1.1 with respect to predicting improved PFS. Meth-NPY is an early indicator of progression, allowing treatment reorientation at an earlier timepoint.Despite several limitations, the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) are still the gold standard in response evaluation of metastatic colorectal cancer (mCRC). The aim of the present study was to investigate hypermethylated  Progression-free survival (PFS) is a clinically relevant and validated endpoint for the effect of first-line treatment in metastatic colorectal cancer (mCRC), independent of subsequent treatment lines. At present, the gold standard for response evaluation is RECIST 1.1  . MoreoveCirculating tumor specific DNA (ctDNA) represents an attractive biomarker in plasma that can be measured as a fraction of circulating total cell free DNA (cfDNA) and has potential for real-time monitoring of tumor status in patients with solid tumors . It may NPY) to be downregulated when the promoter region of the gene was hypermethylated [NPY encodes the neurotransmitter neuropeptide Y (NPY), which is involved in cell invasion and proliferation [NPY, for CRC diagnosis [NPY promotor region (meth-NPY) has, thus, been suggested as a promising biomarker in CRC [Epigenetic alterations, i.e., aberrant methylation of DNA, play an important role in carcinogenesis and represent another approach for measuring ctDNA. Changes in the methylation of the promoter region of a gene affect the transcription of the gene; hypomethylation usually increases gene transcription, while hypermethylation inhibits transcription . The silthylated . NPY encferation ,14. It hferation . Roperchiagnosis , while Giagnosis . Hypermer in CRC ,15,16, bMeth-ctDNA can be measured with high sensitivity by droplet digital polymerase chain reaction (ddPCR) allowing for the detection of very small fractions of meth-ctDNA ,18.Our group previously presented survival data from the randomized, double-blind, placebo-controlled phase II FOLFOXIRI-Toco trial investigating 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI) combined with δ-tocotrienol or placebo as supportive treatment in first-line treatment of mCRC . The aimClinicalTrials.gov (NCT02705300). Reporting of the study followed the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) criteria [The study was approved by The Regional Committee on Health Research Ethics for Southern Denmark S-20150185) and the Danish Medicines Agency (2015122439), and it was prospectively registered with 150185 anBetween May 2016 and December 2018, 70 patients with unresectable mCRC were included in the FOLFOXIRI-Toco study for first-line treatment at the Danish Colorectal Cancer Center South, Vejle Hospital, Denmark. All patients were treated with FOLFOXIRI and were randomized to receiving δ-tocotrienol or placebo. The inclusion criteria were adenocarcinoma in the colon or rectum, age 18–75 years, ECOG performance status (PS) 0–1 (patients above 70 years were eligible if PS = 0), and evaluable but not necessarily measurable disease according to RECIST 1.1. Previous adjuvant chemotherapy for radical treatment of stage II or III colorectal cancer was allowed in patients proven disease-free for more than 6 months. Treatment was evaluated every 8 weeks by CT scan according to RECIST 1.1. Treatment was discontinued in the case of progressive disease (PD), unacceptable toxicity, patient request, or death, or as decided by the treating physician.g for 10 min within 4 h of sampling and stored at −80 °C until analysis.Plasma samples were collected at baseline, before every treatment cycle (every 2 weeks), and at every follow-up visit  or until progression. Firstly, 18 mL of blood was drawn in EDTA tubes for marker analysis. The samples were centrifuged at 2000× g for 10 min. DNA was purified from 4 mL of plasma using the QIAsymphony circulating DNA kit , and cysteine-rich polycomb-like protein 1 (CPP1) DNA fragments were added as an exogenous internal control [Meth-NPY was defined as DNA with methylation of the NPY gene promoter . Prior t control . Then, 2 control  was perf control . The uppAt baseline, patients were divided into two subgroups: above and below the median of meth-NPY. After the first treatment cycle, patients were further divided into two subgroups according to meth-NPY response: responders and non-responders. Response rate according to meth-NPY was defined as the fraction of patients converting from a baseline detectable level to an undetectable level after the first treatment cycle. The undetectable level comprised zero values and those with the lower 95% CI including zero.During treatment and follow-up, a value with no overlap between the 95% CI of the current and preceding measurements was considered a significant increase in meth-NPY.N = 5).Patients with response according to RECIST 1.1 included those with partial response (>30% reduction in target lesions) and complete response. For patients undergoing surgery, the best response on imaging prior to surgery was registered. Patients with non-evaluable or unknown response were not included in the response analysis  tool ,24 hosteBaseline characteristics were compared using Fisher’s exact test and the Wilcoxon Mann–Whitney test for binary and continuous variables, respectively.Response rate based on meth-NPY after one treatment cycle was correlated with survival and response according to RECIST 1.1. Survival analyses were calculated from the day of the first treatment to the date of progression or death of any cause or censored at surgery or last hospital contact (PFS) and to the date of death (OS), respectively. Follow-up was censored at the time of data work-up (November 2021). Median follow-up was calculated using the inverse Kaplan–Meier method. Kaplan–Meier curves illustrating survival were compared with the log-rank test, and a Cox regression model was used to estimate hazard ratios (HRs).p < 0.20 in the univariate Cox regression analysis.Multivariate survival analysis was performed using the Cox regression model with the proportional hazard assumption tested. Together with age, gender, and randomization to either δ-tocotrienol or placebo, the parameters entered in the multivariate Cox regression were variables with p-values were two-sided, and p-values <0.05 were considered statistically significant. Statistical analyses were performed using STATA 17.0 Stata Corp. College Station, TX, USA.All reported Data cutoff for updated survival analyses was performed on 1 November 2021. The median follow-up time was 48.5 months.Sixty-eight of 70 patients had blood sampled at baseline, and 66 of 69 had blood sampled after the first treatment cycle. Sixty-five patients were evaluable for response by RECIST 1.1. During chemotherapy, 16 patients underwent surgery for metastases. Nine patients had partial response on the last scan before surgery, and seven had stable disease.Seven (10.3%) and 19 (28.8%) patients had an undetectable level of meth-NPY at baseline and after the first treatment cycle, respectively. Six patients had an undetectable level of meth-NPY at both timepoints. Baseline characteristics according to meth-NPY at baseline are shown in The median level of meth-NPY was 15.45% (range 0–97.5%) at baseline and 1.15% (range 0–52%) after the first treatment cycle.p = 0.05, HR 2.3, 95% CI 0.97–5.65). Patients with undetectable and detectable meth-NPY at baseline had a median OS of 34.5 months (95% CI 16.7–NR) and 20.6 months (95% CI 15.0–32.5), respectively . Comparing patients with meth-NPY below and above the median at baseline resulted in a significant difference in PFS, 11.4 vs. 7.6 months . There was no significant difference in OS  (The seven patients with undetectable meth-NPY at baseline had a median PFS of 11.9 months (95% CI 7.3–not reached (NR)) compared to 8.6 months in the 61 patients with detectable meth-NPY (95% CI 7.4–10.0) (log-rank 82–2.43) a,b.p = 0.02, HR 0.43, 95% CI 0.21–0.88), , (p = 0.15) and RECIST 1.1 (p = 0.15) , . Patient5–1.21), . No stat = 0.15) .p = 0.008) and PS  remained prognostic factors  and 34 (49%) patients progressed. One patient (1%) died during the first treatment cycle, and two patients (3%) transferred their follow-up to another hospital and were censored before progression.Ten patients had undetectable meth-NPY at progression. The blood sample at progression was missed in one patient. Calculation of lead time was performed in 51 patients with a median of 49 days (range 0–155). Patients progressing during chemotherapy  had a median lead time of 49 days (range 0–119) compared to 51 days (range 0–155) in patients progressing in the follow-up period.Early meth-NPY response has potential as a minimally invasive tool to predict treatment effect, and, in this study of first-line treatment in mCRC, early meth-NPY response performed better in predicting PFS than response according to RECIST 1.1.The latest RECIST guideline (revised RECIST 1.1) was published in 2008, and, despite several limitations, it is still the gold standard of evaluation in mCRC . PFS is WIF1 and NPY) were analyzed in 73 patients [Several studies are pointing toward ctDNA as a reliable tool in evaluation of treatment effect in mCRC. Our group previously investigated meth-NPY in 123 patients with mCRC receiving first line treatment with 5-FU, oxaliplatin, and bevacizumab, divided into two subgroups . Of notepatients . In evalMeth-NPY response challenges the RECIST guideline as to evaluation of patients treated with systemic therapy. In our study, an early NPY response 2 weeks after the first treatment was superior to RECIST 1.1. in predicting PFS. The meth-NPY response rate after one treatment cycle was 31% compared to the objective response rate of 57%, but the higher objective response rate did not reflect a significant difference in PFS. This places meth-NPY as a promising early marker compared to imaging where best response is often achieved up to several months after treatment start. Early meth-NPY response gives an opportunity for drug evaluation in trials earlier than today’s standards. In the multivariate analysis, meth-NPY response was the strongest variable, and randomization between δ-tocotrienol or placebo was not prognostic.The hypothesis of discovering treatment resistance earlier in the blood than by imaging was proven by a significant increase in meth-NPY in plasma with a median of 49 days before progression was found on the conventional CT scan. Other studies from our center support this finding in which mCRC patients receiving standard first-line treatment  or last-In many patients, chemotherapy resistance is visible in the blood earlier than on imaging. Some patients are, therefore, potentially exposed to ineffective, harmful treatment, and their access to other potentially effective treatments is delayed. This suggests serial assessment of meth-ctDNA as a supplement to imaging to improve monitoring in mCRC by applying it between radiological assessments as suggested by Barault et al. . The groUsing immune checkpoint inhibitors in the treatment of a subgroup of patients with mCRC has added another challenge to response evaluation by imaging due to pseudo-progression ,31, but The reporting on ctDNA monitoring of treatment effect in metastatic cancer has recently been discussed . CurrentMutated ctDNA is broadly investigated in mCRC and holds promise as an early marker of treatment effect in many patients, but the most common mutations are only found in about half of mCRC patients, leaving a subgroup in which another type of biomarker is required. Searching for random mutations by whole-genome sequencing or next-generation sequencing in these patients is a time-consuming and expensive procedure, and it is not available in every laboratory. Meth-ctDNA can be detected by ddPCR, a fast and less expensive procedure.http://clinicaltrials.gov/ct2/show/NCT04680260 (accessed on 25 October 2021)).In relation to applying meth-NPY as a universal biomarker in mCRC, attention must be paid to the fact that, in 10.3% of the patients in our study, meth-NPY was undetectable at baseline. Other studies found the marker to be undetectable in 14–20% of their populations at baseline ,26. BaraThe strengths of the study include the prospective design providing reliable data quality and the high internal validity offered by a single-center study. ctDNA was analyzed batch-wise to reduce variation, and the universal characteristic of methylated ctDNA reduces the need for several mutation analyses. Our study is limited by the relatively small number of patients, the retrospective analysis, and the fact that there is no validation cohort, which impairs the generalization of our conclusions. Furthermore, the potential biological variation in measurements was not taken into account .Meth-NPY as an early measure of response to first-line treatment of mCRC proved to be superior to RECIST 1.1 with respect to predicting PFS. Meth-NPY response is an early indicator of clinical benefit and a promising endpoint in clinical trials. A significant increase in meth-NPY during treatment or follow-up is an early indicator of progression allowing treatment reorientation at an earlier timepoint.


Penaeus vannamei) have been vulnerable to the stress induced by different aquaculture operations such as capture, handling, and transportation. In this study, we developed a novel clove oil-nanostructured lipid carrier (CO-NLC) to enhance the water-soluble capability and improve its anesthetic potential in whiteleg shrimp. The physicochemical characteristics, stability, and drug release capacity were assessed in vitro. The anesthetic effect and biodistribution were fully investigated in the shrimp body as well as the acute multiple-dose toxicity study. The average particle size, polydispersity index, and zeta potential value of the CO-NLCs were 175 nm, 0.12, and −48.37 mV, respectively, with a spherical shape that was stable for up to 3 months of storage. The average encapsulation efficiency of the CO-NLCs was 88.55%. In addition, the CO-NLCs were able to release 20% of eugenol after 2 h, which was lower than the standard (STD)-CO. The CO-NLC at 50 ppm observed the lowest anesthesia (2.2 min), the fastest recovery time (3.3 min), and the most rapid clearance (30 min) in shrimp body biodistribution. The results suggest that the CO-NLC could be a potent alternative nanodelivery platform for increasing the anesthetic activity of clove oil in whiteleg shrimp (P. vannamei).Whiteleg shrimp ( In 2020, the global production of whiteleg shrimp was 5.81 million tons [The whiteleg shrimp ion tons . Stress ion tons ,5. Furthion tons , which cion tons .Mentha piperita [M. spicata [Eucalyptus sp., and Origanum sp. [Syzygium aromaticum [M. piperita (Europe and the Middle East), M. spicata (Europe and Asia), Origanum sp. (south-west Asia), S. aromaticum (south-east Asia), and Eucalyptus sp.  are available in their native areas and have been adapted to many regions of the world [Most operations in shrimp farming are conducted without anesthetics, but live transportation of shrimp can be challenging due to their quick movement, cannibalistic behavior, and sharp rostrum . Furtherpiperita , M. spic spicata , Eucalypanum sp. , and Syzomaticum ,15 have he world . (Syzygium aromaticum) is preferable to synthetics since it is more environmentally friendly and cost-effective [P. vannamei [Macrobrachium rosenbergii [Palaemonetes sinensis [P. monodon [The essential oil extracted from cloves ffective . The actffective . Previouvannamei , Macrobrenbergii , Palaemosinensis  and P. m monodon .Chemical anesthetics have limited application owing to safety issues in human beings and fish . In contIn this study, we aimed to determine the physicochemical characterization of NLCs followed by in vitro drug release. Furthermore, the appropriate concentration of CO-NLCs for anesthetic induction and recovery time, as well as the toxicity and biodistribution of CO-NLCs in shrimp, was optimized.The animals and protocol of this study were officially approved by the Institutional Animal Care and Use Committee of the Faculty of Veterinary Science, Chulalongkorn University (IACUC: 2031018). All procedures were carried out according to the university’s guidelines and regulations, as well as policies governing biosafety procedures. Pure essential clove oil was purchased from Thai-China Flavors and Fragrances Industry Co., Ltd. . Polysorbate 20, sorbitan oleate 80, and glycerol were from Croda, Thailand. Cetearyl alcohol and coco-glucoside were supplied by Chemico Inter Corporation, Thailand.Prior to use as an anesthetic, clove oil should be mixed with ethanol, which may be detrimental to aquatic animals after discharging into water bodies. Moreover, it could be a skin allergy to unskilled labor. Therefore, we proposed a safe, cost-effective, and easy-to-use form of clove essential oil by converting the poorly water-soluble CO into a soluble novel nanodelivery platform of herbal anesthetics in whiteleg shrimp . To achiThe formulation of CO-NLCs was prepared by the high-speed homogenization method  with sliThe formulation of CO-NLCs was evaluated for particle size, polydispersity index (PDI), and zeta potential by dynamic light scattering (DLS) . The measurements were performed in triplicate with particle suspensions diluted in distilled water (a ratio of 1:100) at room temperature. The morphology of CO-NLCs was examined by transmission electron microscopy (TEM) . Before the analysis, the CO-NLCs were dispersed in distilled water. A drop of nanoparticle suspension was placed on a carbon-coated copper grid followed by drying. Images were taken at 200000× magnification. The accelerated stability study was carried out to determine the shelf life of the CO-NLCs . The samThe HPLC technique was used to determine the encapsulation efficiency (EE) and release profile of eugenol . The drui represented the initial concentration of eugenol in the CO-NLCs; Cf represented the concentration of unencapsulated eugenol.CP. vannamei) with an average weight of 3 g were purchased from a commercial shrimp farm in Samut Songkhram, Thailand and transferred to the aquaculture laboratory. Shrimps were distributed equally into 30 tanks (300-L capacity) and fed twice a day with commercial feed  at the rate of 2% body weight. During the trial, water quality parameters such as temperature (28 °C), pH (8.2), DO (6 mg/mL), and salinity (20 ppt) were monitored.The whiteleg shrimp  containing different concentrations of the STD-CO and CO-NLCs  in triplicates. The classification of anesthesia and recovery stages are represented in The induction and recovery times were recorded by one observer per stage. The treatment was stopped after the shrimp showed a complete loss of equilibrium (induction time stage 2). The shrimp were transferred to the recovery tank and observed until full recovery in stage 2.The acute toxicity test was conducted by following the method of Cansian, et al.  with sliIn this experiment, we investigated the biodistribution of fluorescently labeled clove oil in shrimp bodies visualized by bioluminescence imaging . Shrimpst-test was used to compare the statistical difference between the means of the STD-CO and CO-NLCs. The Pearson’s correlation coefficients (r) were obtained for different concentrations or times of the STD-CO or CO-NLCs. LC50 was calculated from Probit analysis. Kaplan–Meier survivorship curves and a log-rank (Mantel–Cox) test were used to compare the groups based on the cumulative survival percentage. A value of p < 0.05 was considered statistically significant.Data were expressed as the mean ± standard deviation (SD) for each group, and the statistical analyses were performed using SPSS (Version 26). The student The clove oil was insoluble in water  and CO-NThe aggregation or accumulation of CO-NLCs were not shown in the images of the transmission electron microscope (TEM). The spherical CO-NLCs A were as(p < 0.05) compared to the initial size and zeta potential in either condition. The CO-NLCs were stable for up to 3 months of storage in different conditions (30 °C/60% RH and 40 °C/75% RH) . The CO-p < 0.01) very strong positive (R2 = 0.915 and 0.926) correlation of releasing ability with time . In contrast, the CO-NLCs were able to sustain the release of eugenol. Only 5% of eugenol was gradually released from the CO-NLCs in the first hour. In addition, less than 20% of eugenol was released and detected after 2 h, which was significantly lower than the STD-CO . The linear regression trendline of drug release appeared between 2 to 8 h duration.The result indicates that the release profiles of the STD-CO exhibited a burst release. Eugenol was immediately released in a few minutes and reached the 30% level within 1 h, which was significantly higher than the CO-NLCs (p < 0.01) strong negative correlation of induction (R2 = −0.706 and −0.852) and a very strong positive correlation of recovery time (R2 = 0.946 and 0.912) with concentrations. Both the STD-CO and CO-NLCs were able to induce anesthesia in shrimp that entered stage 2 without complications. The 20 ppm was the lowest concentration of the STD-CO and CO-NLCs that allowed shrimp to reach stage 2  with 6.30 min. However, the induction time of the STD-CO treatment decreased to 12  and 5 min  at 30 and 40 ppm concentration, respectively. Furthermore, for medium concentrations (30–70 ppm), the CO-NLCs had a significantly (p < 0.05) shorter duration of induction time than the STD-CO. The higher concentrations (100–200 ppm) of the STD-CO and CO-NLCs demonstrated rapid anesthetic effects on shrimp within 0.85–3 min after immersion in the CO-contained tanks.The STD-CO and CO-NLCs showed a significantly ( stage 2 A. Interep > 0.05) in recovery time for medium concentrations (30–70 ppm) between the STD-CO and CO-NLCs. The higher concentration of the STD-CO resulted in a longer recovery duration than the CO-NLCs. With an increasing concentration of clove oil, the duration required to achieve sedation and anesthesia decreased, and the recovery times increased for the STD-CO shrimp in contrast to the CO-NLCs.The STD-CO and CO-NLCs treatments with 20 ppm concentrations did not differ in the recovery times (3.50 and 3 min) for shrimps from the complete loss of equilibrium stage B. Similap < 0.01) strong (R2 = −0.791) and moderate (R2 = −0.436) negative correlation with concentrations, indicating the higher concentrations can increase the mortality rate. The median lethal concentrations (LC50) of the STD-CO and CO-NLC calculated from the Probit regression analysis were 143.8 ± 2 ppm and 353.1 ± 16.4 ppm, respectively. The log-rank (Mantel-Cox) test from the Kaplan–Meier analysis found statistically significant differences in the cumulative survival percentage for all the concentrations of the STD-CO (X2(19) = 473.373, p < 0.01) and CO-NLC (X2(19) = 343.881, p < 0.01)  A,B. The  < 0.01) A. Moreov < 0.01) B.The biodistribution of the STD-CO and CO-NLCs of the shrimp was examined through fluorescence signal imaging on the shrimp body at different times after full recovery. The fluorescence intensity was represented by a multicolor range from green (low intensity of CO) to red (high intensity of CO). From whole-body imaging , the CO-The quantification of fluorescence intensity of whole-body imaging using ImageJ software confirmed the presence of CO accumulation. The results showed that the excretion of the CO-NLCs was faster than the STD-CO . In 30 mThe use of anesthetics such as MS-222 and Aqui-SM in aquaculture has been approved for human consumption in several countries . The conIn the present study, we investigated the physicochemical characterization of the CO-NLCs, a new lipid nanoparticle-carrier system of clove oil that is strong enough to be used as an alternative platform for anesthetic agents in shrimp. In general, STD-CO is insoluble in water. Therefore, STD-CO needs to be dissolved and diluted in ethanol before it can be used as an anesthetic. In drug-delivery systems, PDI values ≤ 0.3  are acceRegarding the release profile pattern, clove oil adsorbed on the surface was responsible for the initial burst of release when it became accessible at the interface, while the embedded clove oil was responsible for the persistent release . The STDPiaractus brachypomus [P. semisulcatus [The biological characteristics of clove oil to be used as an anesthetic in immersion mode have also been assessed in this study. The STD-CO showed that the increased exposure period of clove oil resulted in a longer recovery time A. Similachypomus . Importachypomus ,44. Coatsulcatus . The extsulcatus ,46, appeA) neurotransmitters and metabotropic glutamate receptors, both of which have been identified in certain invertebrates [A receptor in the central nervous system [A receptors’ response. Thus, additional study is needed to determine how CO-NLCs activate the GABAA receptor response.The mechanisms of CO-NLCs in shrimps are not well known. The anesthetic response could be regulated by γ-Aminobutyric acid type A  than those of the STD-CO (144 ppm) concerning the biodistribution and excretion results , the recovery time (3.3 min) and the rapid clearance (30 min) in shrimp body biodistribution. Our finding highlights a potential opportunity for NLC as a potent alternative nanodelivery platform which can be suitable for the enhanced anesthetic activity of clove oil in whiteleg shrimp. Further studies are needed to determine how the CO-NLCs activate the GABA


Furthermore, while the method is simple to perform, its reproducibility is poor.The traditional method for assessing the capacity of a microorganism to produce biofilm is generally a static Staphylococcus aureus and Candida albicans.We evaluated whether matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) could make it possible to differentiate between high-and low-biofilm-producing microorganisms on 24-h cultures of S. aureus and 91 strains of C. albicans obtained from the blood cultures of patients with bacteremia/candidemia. We tested biofilm production using the CV binding assay as the gold standard to classify strains as low or high biofilm producers. We then applied MALDI-TOF MS to create a machine learning–based predictive model using 40 strains of S. aureus and C. albicans, each with extreme absorbance values, and validated this approach with the remaining 117 and 51 strains using the random forest algorithm and the support vector machine algorithm, respectively.We included 157 strains of S. aureus strains (95/117) and 74.5% of the C. albicans strains (38/51) used for validation were correctly categorized, respectively, as low and high-biofilm-producing.Overall, 81.2% of the S. aureus and C. albicans to form biofilm.Classification based on MALDI-TOF MS protein spectra enables us to predict acceptable information about the capacity of 24-h cultures of  S. aureus and C. albicans to form biofilm.We provide a new application of MALDI-TOF MS protein spectra that enables us to predict acceptable information about the capacity of 24-h cultures of in vitro model used  is based on the detection of the protein profile of the microorganism through its species-specific spectra . The incThe present study aims to determine the ability of MALDI-TOF MS to classify strains according to their capacity for biofilm production (high or low) compared with traditional biofilm determination based on CV staining.Staphylococcus aureus . The inoculum was then incubated with orbital shaking (165 rpm) at 37°C for 24 h. The tubes were then centrifuged at 1,500 rpm for 5 min and washed three times with PBS (pH = 7.4). We used the resulting suspension to prepare a suspension containing 108 cfu/mL (0.5 McFarland) for S. aureus and 106 cfu/mL (0.35 McFarland) for C. albicans. The polystyrene 96-well plate  was inoculated with 100 μL of each suspension and incubated at 37°C for 24 h. The medium was then removed from the wells, and the plate was washed 3 times with PBS (pH = 7.4) and left to dry. Finally, CV staining was performed to measure biomass in a spectrophotometer , with absorbance read at 550 nm (We tested biofilm production using the static 96-well plate method (gold-standard). An inoculum was prepared from 24-h cultures by inoculating several colonies in 50 mL of enrichment medium in Falcon tubes (tryptic soy broth for t 550 nm .S. aureus were classified as high (≥1.292), moderate (0.530–1.291), and low (<0.530); those for C. albicans were also classified as high (≥1.428), moderate (0.922–1.427), and low (<0.922).We classified strains into tertiles in ascending order according to their absorbance values obtained by the CV binding assay . Thus, aS. aureus and Candida Chrom-agar  for C. albicans) based on a previous pilot study using 40 strains of S. aureus in which strains were equally classified using either 24-h-cultured or resuspended sessile cells grown in wells once biofilm had formed (data not shown). A colony of each strain was spotted on two positions of the MALDI-TOF MS plate and covered with 1 μL of 100% formic acid in order to lyse the cells and allow them to dry at room temperature. Then, 1 μL of organic matrix  was added and allowed to dry. The plate was analyzed using an MBT Smart MALDI Biotyper  in the range between 2,000 and 20,000 Daltons.MALDI-TOF MS was performed on 24-h-cultured isolates  or low-biofilm-producing (n = 20) to generate predictive models. We applied the machine learning supervised algorithms partial least squares-discriminant analysis (PLS-DA) and random forest plot (RF). In order to validate the model, we subsequently included the remaining 117 S. aureus strains (58 high-and 59 low-biofilm-producing) and 51 C. albicans strains (25 high-and 26 low-biofilm-producing). ROC curves representing the probability of identification obtained for each algorithm were created.The MALDI-TOF MS plate was read twice, and spectra were acquired using default settings. They were then processed and further analyzed using Clover MS Data Analysis software  in the following steps: (1) Variance stabilization; (2) Smoothing by Savitzky–Golay filter with window length = 11 and polynomial order = 3; (3) Baseline subtraction (top-hat filter = 0.02); and (4) Normalization by total ion current. To determine agreement between the gold standard and MALDI-TOF MS, we selected 40 strains of each species classified according to the above tertiles as high-biofilm-producing , violet dots represent the high-biofilm-producing strains (n = 20), and red dots represent those used to validate the model (117 high-and low-biofilm-producing strains). The same number of strains in each category (low-or high-producing) was misidentified by PLS-DA, and similar numbers were observed for RF . k-fold cross-validation yielded higher accuracy with the RF algorithm 97.1%, . Howeverd for RF . Moreoved for RF . Misidend for RF .We obtained a total of 80 spectra from the 40 strains selected. The RF algorithm yielded a k-fold cross validation accuracy of 92.5% . For theS. aureus. In contrast, the correlation for C. albicans was moderate.We observed a good correlation between MALDI-TOF MS and the multi-well plate gold standard for classifying high-and low-biofilm-producing strains of Since the ability to form biofilm can be an indicator of pathogenicity and is widely used in clinical studies, it would be interesting for laboratory results to become available quickly . HoweverS. aureus and C. albicans yielded a high correlation index with the 40 strains used to create the model. However, when the models were validated with the remaining strains, the correlation weakened for the RF algorithm. By contrast, PLS-DA, which was the only linear algorithm applied, yielded the best classification results for both species tested, correctly identifying more than 80% of S. aureus strains. Likewise, comparison of the results obtained for S. aureus with those obtained for C. albicans revealed that the former were even better, as the strains were better categorized, probably because the selected C. albicans strains classified as high biofilm producers did not show high CV absorbance values (unlike S. aureus) and may be closer to the “moderate” than to the “high” category. Moreover, since the misidentified strains of C. albicans had absorbance values on the borderline of high-biofilm-producing strains, they were probably considered low producers by the model. The addition of a higher number of strains with absorbance values covering the complete range could improve the model in the future.Therefore, alternatives are needed to determine whether a strain is high-or low-biofilm-producing in a more rapid and reproducible manner. In our study, we assessed whether MALDI-TOF MS, which has been shown to be highly capable of identifying for intra-and interspecies differences associated with various factors, could be useful for differentiating between strains according to their ability to produce biofilm. Therefore, we chose the two most virulent species with the worst prognosis for patients, as it is important to clarify the association between biofilm production and patient prognosis . SpecifiStaphylococcus strains or yeasts. In the first, MALDI-TOF MS was used to predict biofilm-forming capacity in only 18 strains of S. epidermidis, revealing a good correlation with respect to CV staining  of which were high-biofilm-producing (CV absorbance >1.17) and in which the authors demonstrated that MALDI-TOF MS was able to differentiate between specific biofilm proteins, subsequently validating the correlation between 4 high-producing strains and 4 low-producing strains  in order to develop a classification model that will be later tested with a different group of data . Non-supervised algorithms  might be preferable to supervised algorithms  because they are available to a high number of researchers. In this case, the compared algorithms are both non-supervised. PLS-DA seems to be the fittest algorithm for the classification of the analyzed data. It can be applied by using the software described in the article  or by programming with python or R.One of the limitations of the study is that this procedure cannot be applied to polimicrobial biofilms, as two different protein spectra would be obtained.S. aureus and C. albicans according to their biofilm production. Future studies should expand the number of strains, including new species and additionally using whole genome sequencing. Moreover, reproducibility testing should be assessed to confirm our results.Our study supports the potential use of MALDI-TOF MS for classification of clinical strains of both The original contributions presented in the study are included in the article/MG and BR-S were responsible for the organization and coordination of the trial. DR-T, MD-N, BR-S, PM, and MG were the chief investigator and responsible for the data analysis. DR-T, MD-N, RD, PE, and JG developed the trial design and data collection. All authors contributed to the writing of the final manuscript.MG was supported by the Miguel Servet Program  from the Health Research Fund (FIS) of the Carlos III Health Institute (ISCIII), Madrid, Spain. MD-N was supported by the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid and Fondo Social Europeo (PEJD-2020-AI_BMD-17971). DR-T was supported by the Instituto de Investigación Sanitaria Gregorio Marañón through its intramural program. BR-S was supported by the Miguel Servet Program . PE (CPII20/00015) is the recipient of a Miguel Servet contract supported by the FIS. JG is a full-time researcher contracted by Fundación para Investigación Sanitaria del Hospital Gregorio Marañón. The study was partially funded by grants from the Fundación Mutua Madrileña (FMM21/01), ISCIII (PI21/00344), and European Regional Development Fund (FEDER) “A way of making Europe.”JG has received funds for participating in educational activities organized on behalf of Gilead, MSD, and Pfizer, and also received research funds from FIS, Gilead, Scynexis, F2G, and Cidara, outside the submitted work.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Salviae miltiorrhizae, one of the most important traditional herbal medicines widely used in several countries for the treatment of cardiovascular diseases. The aim of this study was to evaluate the in vitro effect of SalB on the expression and the activity of matrix metalloproteinase 9 (MMP-9), a zinc-dependent proteolytic enzyme, in human MDA-MB-231 breast cancer cells. This cellular model is characterized by a marked invasive phenotype, supported by a high constitutive expression of MMPs, especially gelatinases. SalB was first of all evaluated by in silico approaches primarily aimed at predicting the main pharmacokinetic parameters. The most favorable interaction between the natural compound and MMP-9 was instead tested by molecular docking analysis that was subsequently verified by an enzymatic inhibition assay. MDA-MB-231 cells were treated with SalB 5 µM and 50 µM for 24 h and 48 h. The conditioned media obtained from treated cells were then analyzed by gelatin zymography and reverse zymography to, respectively, evaluate the MMP-9 activity and the presence of TIMP-1. The expression of the enzyme was then evaluated by Western blot on conditioned media and by analysis of transcripts through reverse transcriptase-polymerase chain reaction (RT-PCR). The in silico approach showed the ability of SalB to interact with the catalytic zinc ion of the enzyme, with a plausible competitive mode of action. The analysis of conditioned culture media showed a reduction in MMP-9 activity and the concomitant decrease in the enzyme concentration, partially confirmed by analysis of transcripts. SalB showed the ability to modulate the function of MMP-9 in MDA-MB-231 cells. To our knowledge, this is the first time in which the role of SalB on MMP-9 in a highly invasive cellular model is investigated. The obtained results impose further and more specific evaluations in order to obtain a better understanding of the biochemical mechanisms that regulate the interaction between this natural compound and the MMP-9.Salvianolic acid B  is a bioactive compound from  The MMP-9/Gelatinase B belongs to the wide family of zinc dependent endopeptidases named matrix metalloproteinases (MMPs). MMPs are involved in the physiological degradation of the extracellular matrix (ECM), a fundamental process for tissue development, morphogenesis, remodeling and repair . Due to Disturbances of this complex network hindering the normal MMPs function are known to be associated with the development of pathological events related to excessive or insufficient ECM turnover: arthritis, wound healing disorders, fibrotic diseases and cancer . Regarding cancer, it is known that the proteolytic activity which contributes to the degradation of ECM and basal membrane represents a key step in cell invasion, metastasis and angiogenesis . MoreoveIn the last decade, great interest was generated in the characterization and use of natural compounds credited with high bioactive potential, including anti-inflammatory, antibacterial, antioxidant, anti-cancer and anti-diabetic properties ,12,13. ISalvia miltiorrhiza, also known as red sage or Chinese sage, is one of the most widely used traditional herb medicines recommended for the treatment of a variety of diseases, such as cardiovascular diseases, hepatitis, hepatocirrhosis, chronic renal failure and dysmenorrhea. In S. miltiorrhiza, two pharmacologically active fractions were identified: lipophilic diterpenoids transhinones and water-soluble phenolic acids , trypan blue, Triton X-100, Tween 20 and type B gelatin were purchased from Sigma-Aldrich Chemical Co. . Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS), penicillin, streptomycin, glutamine and trypsin were purchased from Euroclone S.p.A. . Human recombinant catalytic domain of MMP-9 was obtained from Vinci Biochem S.r.l. . All other chemicals were reagent grade. Stock solution of SalB (50 mM) was prepared in DMSO and stored in the dark at −20 °C. http://www.swissadme.ch/index.php, accessed on 16 November 2022) . Such parameters were then used to draw a secondary plot for the determination of the apparent inhibition constant (Ki). The apparent Michaelis constant (K2 at 37 °C. Cellular viability was determined by trypan blue exclusion assay.The The MDA-MB-231 human breast cancer cells were obtained from the American Type Culture Collection (ATCC) and maintained in exponential growth in DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, 100 mg/mL streptomycin and 2 mM glutamine, and kept in a humidified atmosphere with 5% COThe concentration range and the timing of the cellular treatment with the SalB have been defined through the MTT colorimetric approach which measures viable cells by assessing the conversion of MTT into formazan crystals by mitochondrial activity . Exponen4 cells/well. After 24 h, the medium was replaced with fresh medium and cells were treated with 5 µM  and 50 µM  of SalB. Control cells (CTR) received DMSO in a concentration equal to that used for the solubilization of Sal-5 and Sal-50. After 24 h and 48 h, conditioned media were collected, aliquoted and stored at −20 °C until analysis. After defining the parameters for cell treatment with SalB, cells were seeded in six well culture dishes at a density of 5 × 102, 200 mM NaCl and 0.02% Brij 35) was performed to allow enzyme renaturation and activity. Gels were then stained in a 0.1% solution of Coomassie Blue R250 in 40% (v/v) methanol and 10% (v/v) acetic acid. This analysis was preceded by preliminary evaluations, in which the supernatant of HT1080 fibrosarcoma cells was used as a reference standard for both MMP-2 and MMP-9, as recommended by Toth et al. [Protein concentration in conditioned media was determined by Bradford protein concentration assay. Volumes of each sample corresponding to 10 µg of total proteins were diluted in a non-reducing sample buffer without heating and resolved by 8% SDS-PAGE containing 0.3 mg/mL type B gelatin. The gels were then incubated for 45 min in a renaturation buffer  to remove SDS. Subsequently, a 24 h incubation in the developing buffer  transfer membranes. The non-specific protein binding site on membranes was blocked by incubation in a solution containing 5% non-fat dry milk  in TBS 0.1% Tween 20 (TBS-T) for 1 h, and then incubated overnight at 4 °C with the primary antibodies for human MMP-9 and TIMP-1 diluted in 1% non-fat dry milk in TBS-T. Membranes were then washed in TBS-T, incubated for 1 h with secondary HRP-conjugated antibody diluted in blocking solution, and the immunoreactive bands were detected by inducing a chemiluminescence reaction through the ECL chemiluminescent reagent . Quantitative analysis of immunoreactive spots was performed by using ImageJ software .Total RNA was extracted from treated cells with EUROGOLD Total RNA Mini kit  and 1 μg of each sample was subjected to retrotranscriptase reaction and PCR amplification in the same tube using the HyperscriptTM One-step RT-PCR Premix  and following manufacturer’s indications. Expression levels of MMP-9 and TIMP-1 were normalized to the expression levels of the housekeeping β-actin gene. PCR was performed by using the following oligonucleotides: MMP-9 , TIMP-1  and β-actin . The RT-PCR products were resolved by 1.5% agarose gel electrophoresis. p < 0.05) and 99% (p < 0.01). Data were statistically analyzed by using SigmaPlot 12.0 software  for Windows operating system. Differences between means were evaluated by Student’s t-test with confidence levels set at 95% (Salviae mitiorrhizae, to modulate the function of MMP-9 in human MDA-MB-231 breast cancer cells was demonstrated in an in vitro model characterized by a high invasive potential. Particularly, we found a marked tendency of the natural compound to interact with the catalytic domain of MMP-9 with the consequent inhibition of its activity, presumably reducing the ability of the enzyme to interact with the substrate. Furthermore, an inhibitory effect on the enzyme expression was observed. From this point of view, more specific studies will have to be conducted in order to characterize the biochemical mechanisms that oversee this phenomenon.In the present research, for the first time to our knowledge, the ability of salvianolic acid B, an active compound of


IntroductionThe cycle threshold (Ct) value in real-time reverse transcription-polymerase chain reaction (RT-PCR) serves as a criterion to diagnose coronavirus disease 2019 (COVID-19) and is inversely proportional to viral load. Levels of inflammatory markers such as aspartate aminotransferase (AST), ferritin, D-dimer, high sensitivity C-reactive protein (hs-CRP), and lactate dehydrogenase (LDH) are used as quantitative measures of COVID-19 severity. We examined the association between these markers and Ct values.MethodologyThis retrospective data analysis included 400 patients with positive RT-PCR results for COVID-19 who were admitted to a tertiary care hospital. Clinical and biochemical data were accessed from the hospital information management system. Associations of clinical parameters and markers of disease severity  with Ct values were assessed.ObservationsLDH, ferritin, D-dimer, and hs-CRP were found to be significantly higher in moderate and severe groups than in the mild COVID-19 group. AST, ferritin, and hs-CRP levels were also significantly higher in severe COVID-19 subjects, compared to moderate COVID-19 subjects. Ct values for the E (envelop) gene and ORF (open reading frame) 1b gene were found to be significantly higher in those with severe COVID-19. Polymorph counts in subjects with Ct values of 25 or higher were significantly increased, compared to those with Ct values under 30. LDH, D-dimer, and hs-CRP levels in subjects with Ct values over 30 were significantly lower than for those with Ct values under 30. Ferritin was the best independent predictor of non-survival in study subjects, with an area under the curve (AUC) of 85.5% . The Ct value for the E gene had an AUC of 75.1%, and the ORF1b gene had an AUC of 64.5%. However, no significant correlation was detected between any parameter and Ct value.ConclusionPolymorph, LDH, ferritin, D-dimer, and hs-CRP levels were significantly elevated in subjects with low E gene Ct values. Also, these subjects were at risk of severe disease and fatality. Ct values for the E gene thus could serve as an early indicator for patients at risk of severe disease and death. The severe acute respiratory syndrome caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents the greatest pandemic of the 21st century. As of July 2022, India stands second only to the United States with regard to diagnosed cases, with 4,35,00,000 cases. Concerning the death toll, India ranks third worldwide [Inflammatory markers such as the serum cytokines lactate dehydrogenase (LDH), ferritin, D-dimer, interleukin-6, and high sensitivity C-reactive protein (hs-CRP) are used as quantitative measures of the severity of COVID-19. CRP is among the first biomarker to be altered in the inflammation process and thus is a potentially useful predictor of disease outcome in COVID-19 . ElevateFew studies have evaluated the association between the clinical severity of COVID-19 and Ct values. Some studies have reported low Ct values in patients with COVID-19 associated with increased levels of inflammatory biomarkers creatinine kinase and high-sensitivity troponin I and lower levels of lymphocyte, T-cell, and serum albumin . Ct valuThis hospital-based retrospective study was conducted in the Department of Biochemistry in collaboration with the Departments of Medicine and Microbiology at All India Institute of Medical Sciences, Nagpur, India, after approval from the institutional ethical committee . Participants included subjects with positive RT-PCR tests who were admitted to the hospital. Only symptomatic subjects were admitted to the hospital and subjects with all grades, viz. mild, moderate, and severe symptoms were included in the study. Individuals with a history of any acute inflammatory or debilitating illness , ongoing chronic illness , or malignancy were excluded.Sample collection and analysis for RT-PCR were conducted as per standardized procedure . ClinicaRoutine biochemical parameters were analyzed on a Cobas® 6000 modular (c501 and e601) biochemistry and immunoassay analyzer using appropriate reagent kits . The estimation of serum hs-CRP was based on a particle-enhanced immunoturbidimetric assay with a biological reference interval of <5 mg/L. Estimation of serum ferritin was based on an electrochemiluminescence immunoassay with a biological reference interval of 30-400 μg/L in 20-60-year-old males and 13-150 μg/L in 17-60-year-old females. Quality control assessment was conducted via an internal procedure using Cobas PreciControl . Plasma D-dimer was estimated on a STAR Max® hemostasis analyzer  and based on an immune-turbidimetric assay with a biological reference interval of <500 ng/mL. Biological reference intervals followed package inserts of the respective diagnostic kits.Data collectionRates of illness severity and death among COVID-19 patients during hospitalization were the main outcome measures of the study. All clinical, laboratory, and outcome data were taken from the hospital management information system services provided by the Centre for Development of Advanced Computing India Ltd. at All India Institute of Medical Sciences, Nagpur. Data included demographics, clinical blood biochemistry, and viral load (Ct) of COVID-19.Statistical analysisThe sample size was finalized at an α error of 5%, power of 80%, and precision of 0.05. The data were analyzed using appropriate statistical methods in IBM SPSS Statistics for Windows, Version 21.0 . Data were analyzed for a test of normality. Continuous variables were expressed as mean ± SD in the parametric distribution of data and as the median and interquartile ranges (25th and 75th percentiles) in non-parametric distributions. Comparisons within groups were performed using Mann-Whitney U or Kruskal-Wallis tests followed by the posthoc Dunn test. Observed frequencies of categorical variables were compared by Chi-square test. The receiver operator characteristic (ROC) curve was plotted to assess the predictive ability of Ct values and biomarkers for survival. Correlation analysis was performed using Spearman’s rank-order correlation analysis. A p-value of <0.05 was considered significant except for the correlation analysis, for which a p-value of 0.01 (two-tailed) was considered significant.Among 400 participants, 274 had mild illness, 78 had moderate illness, and 48 had severe illness based on established criteria . During The median age of subjects with moderate and severe disease was found to be significantly higher than subjects with mild disease. Study groups were found to be matched for gender distribution (p=0.257). Symptoms of dry cough p<0.0001), breathlessness (<0.0001), fatigue (p<0.0001), loose motions (p<0.0001), and comorbidities such as hypertension (p<0.0001), diabetes mellitus (p<0.0001), and ischemic heart disease (p<0.0001) were found to be significantly higher in frequency in subjects with moderate or severe disease compared to those with mild disease. However, symptoms such as running nose (p=0.008), anosmia (p<0.0001), and ageusia (p=0.007) were associated with mild disease. AST (p<0.0001), LDH, ferritin, D-dimer, and CRP were found to be significantly different amongst mild, moderate and severe COVID-19 subjects . On further post hoc analysis, LDH, ferritin, D-dimer, and hs-CRP were found to be significantly higher in moderate and severe groups compared to the mild COVID-19 group. AST, ferritin, and hs-CRP were also found to be up significantly higher in severe COVID-19 subjects compared to moderate COVID-19 subjects. Ct values for E gene as well as that for ORF 1b gene were found to be significantly higher in severe COVID-19 groups . For each gene group, the Ct subgroups were well-matched for age and gender. For the E gene group, significant differences were noted in levels of total leucocytes (p=0.040), polymorphs (p=0.001), LDH (p<0.0001), ferritin (p<0.0001), D dimer (p<0.0001), and CRP (p<0.0001). The frequency of comorbidities was found to be associated with low Ct value (p<0.0001), severe COVID-19 (p<0.0001), and non-survival (p=0.001). A posthoc analysis showed the polymorph counts in subjects with Ct values of 25 or lower to be significantly higher than in those with Ct values over 30. Also, LDH, D-dimer, and CRP levels in subjects with Ct values over 30 were significantly lower than in those with Ct values under 30. Figure Ferritin was found to be the best independent predictor of non-survival in study subjects . The Ct value for the E gene had an AUC of 75.1% and that of the ORF 1b gene was 64.5%. Table Correlation analysis of various parameters of disease severity with Ct values was performed using Spearman’s rank-order correlation. None of the parameters was found to be correlated with Ct values, regardless of survival or severity subgroup Table .The present study aimed to evaluate clinical and biochemical markers associated with COVID-19 and their relationship with Ct values, which are inversely related to viral RNA load. Disease severity in the study population was categorized as mild, moderate, or severe according to established criteria based on respiratory status and oxygen saturation . We obseClinical correlation of D-dimer with pro-inflammatory cytokines has been observed in critically ill patients with COVID-19 . D-dimerThe RT-PCR Ct value and the SARS-CoV-2 virus load are inversely proportional to each other, thereby indirectly reflecting the severity of infection . Our resScola et al. found that a lower Ct value in severe COVID-19 was associated with a high neutrophil count , which wIn our study, Ct values were significantly lower in subjects with severe disease and in non-survivors, compared to their respective counterparts. It has been reported previously that low Ct values are often associated with severe disease and longer viral persistence. A high viral load may increase the severity of the disease and the chances of death ,27. In aWe found ferritin to be the best independent predictor of non-survival in study subjects . Ahmad et al. have also reported ferritin as a predictor of mortality (AUC 69%) . Ct valuDespite findings of significant differences regarding various biochemical parameters between subjects with different Ct values, our study failed to find any significant correlation between the studied parameters and Ct values in terms of severity and survival. Due to the retrospective study design and functional problems, we could not evaluate potential confounders such as virus strain or variations in RT-PCR techniques. We also estimated only the relationships associated with the Ct value for the E gene and ORF1b gene and not other genes used in RT-PCR studies around the world for diagnosis of COVID-19. Thus, our study has relevance only in the context of these genes. Nevertheless, our study offers novel insight into the utility of Ct value for establishing disease severity and mortality in COVID-19. These listed points may be deemed as limitations to our study and the findings must be interpreted in light of these factors.We conclude that subjects with lower Ct values for the E and ORF1b genes tend to have severe disease and are more susceptible to fatality. Polymorphs and biochemical indicators of disease severity  were significantly elevated in subjects with lower Ct values for the E gene, but these parameters did not show any correlation with Ct values. Ct values for the ORF1b gene showed no association with any indicator of disease severity. RT-PCR is one of the first investigations to be performed in COVID-19 diagnosis, and the patient’s Ct value is usually known at the time of admission. This information, thus, can serve as evidence to use Ct value as an early and relevant predictor of severity and outcome and can be helpful for the treating clinician. We recommend further investigation of the associations between these parameters and Ct value for other genes used in the diagnosis of COVID-19 using a prospective study design.


P = 0.032; HR = 0.70; 95% CI, 0.50–0.97). Incidence of treatment-related adverse events of grade 3–5 was lower with sintilimab than with chemotherapy (20.2 vs. 39.1%). Patients with high T-cell receptor (TCR) clonality and low molecular tumor burden index (mTBI) showed the longest median OS (15.0 months). Patients with NLR < 3 at 6 weeks post-treatment had a significantly prolonged median OS (16.6 months) compared with NLR ≥ 3. The results demonstrate a significant improvement in OS of sintilimab compared to chemotherapy as second-line treatment for advanced or metastatic ESCC.This randomized, open-label, multi-center phase 2 study (NCT03116152) assessed sintilimab, a PD-1 inhibitor, versus chemotherapy in patients with esophageal squamous cell carcinoma after first-line chemotherapy. The primary endpoint was overall survival (OS), while exploratory endpoint was the association of biomarkers with efficacy. The median OS in the sintilimab group was significantly improved compared with the chemotherapy group (median OS 7.2 vs.6.2 months;  Patients with advanced esophageal cancer have poor prognosis and limited treatment options. This randomized, phase II trial compares the efficacy and safety of the anti-PD-1 antibody sintilimab versus chemotherapy in Chinese patients with esophageal squamous cell carcinoma after first-line therapy More than half of global ESSC cases occur in China3. The patients with ESCC have a poor prognosis with a 5-year overall survival rate ≤15%4. As the second-line therapy, single-agent chemotherapy is an established option with limited clinical benefit and common toxicities11. Since 2019, several PD-1 inhibitors are approved as second-line therapy for ESCC14. Currently the most prominent biomarker for PD-1/PD-L1 treatment options is PD-L1 expression, but this biomarker remains to be imperfect in identifying benefitting ESCC patients. It is therefore of greatest importance to identify alternative biomarkers.Esophageal squamous-cell carcinoma (ESSC) is the predominant subtype of esophageal cancer in Asian populations, accounting for 90% of cases18.Sintilimab is a humanized, monoclonal antibody against PD-1 that has been approved by NMPA for the monotherapy of relapsed or refractory classical Hodgkin lymphoma, the first-line treatment of nonsquamous and squamous NSCLC in combination with chemotherapy, and the first-line treatment of hepatocellular carcinoma in combination with bevacizumabHere, we conducted the ORIENT-2 study to compare the efficacy and safety of sintilimab vs. chemotherapy as second-line treatment for advanced or metastatic ESCC. In addition, it was expected to observe the alternative predictive biomarkers for these patients treated with sintilimab through neutrophil-to-lymphocyte ratio (NLR), RNA sequencing (RNA-seq) and circulating-tumor DNA (ctDNA).n = 95 per group, Fig. Between May 16, 2017 and August 30, 2018, 253 patients were screened and 190 eligible patients were randomly assigned to receive either sintilimab or chemotherapy . Meanwhile, a delayed separation of the survival curves was observed , , and , indicating the delayed effect of the significant OS benefit with sintilimab over chemo. It was thus assumed that sintilimab did not increase the risk of death if used at an earlier time according to the Fleming–Harrington  (P = 0.242) , the median duration of follow-up was 7.2 months  for the sintilimab group and 6.2 months  for the chemo group. Overall, 69 (72.6%) patients in the sintilimab group and 81 (85.3%) patients in the chemo group had death. The median overall survival (OS) was 7.2 months  in the sintilimab group compared with 6.2 months  in the chemo group  Table . The est2) Table . MoreoveP = 0.979; stratified HR = 1.00; 95% CI, 0.72–1.39). The median PFS was 1.6 months  in the sintilimab group and 2.9 months  in the chemo group  patients in the sintilimab group and 73 (76.8%) in the chemo group had progressive disease or death. However, the difference in median progression-free survival (PFS) per RECIST v1.1 was not significant , whereas treatment was discontinued for 28 patients (noncontinuous subgroup). A post hoc analysis of OS in the above subgroups indicated that the median OS of patients with continuous treatment was dramatically improved over those with discontinued treatment . However, both groups showed a similar disease-control rate (DCR)  in the sintilimab group  was achieved in comparison with the chemo group . The median duration of response (DOR) of patients in the sintilimab group was longer than the chemo group : age ≤65 years, male, previous treatment with paclitaxel, Eastern Cooperative Oncology Group performance-status (ECOG PS) score of 1, and current smokers Fig. . HoweverHealth-related quality of life was assessed in 87 patients in the sintilimab group and 84 patients in the chemo group at baseline. Based on the change from baseline, patients in the sintilimab group presented with better health-related quality of life from patients in the chemo group Table .The median duration of treatment with sintilimab, paclitaxel, and irinotecan was 11.9 , 11.9 , and 7.0  weeks, respectively. The median relative dose intensity was 100.0% , 96.5% , and 98.2% , respectively.Treatment-emergent adverse events (TEAEs) of any grade occurred in 88 93.6%) patients with sintilimab, and 81 (93.1%) patients with chemo. Treatment-related adverse events (TRAEs) were reported in 54.3% of patients in the sintilimab group and 90.8% of patients in the chemo group  patients in the sintilimab group experienced immune-related adverse events (irAEs), with pulmonary inflammation (9.6%) and hypothyroidism (8.5%) occurring most frequently  and PFS  compared with those with a high NLR at baseline. In total, 65 patients had evaluable NLR at 6 weeks post treatment. Notably, the survival benefit was also significant in patients with low versus high NLR, with a median OS of 16.6 vs. 6.2 months  and a median PFS of 4.3 vs. 2.3 months  had qualifiable RNA-seq data for downstream analysis from tumor tissue. We analyzed an outcome association of 28 immune-cell populations and overall, only few potential positive correlations of immune-cell infiltrations with PFS but not OS could be observed. Only a significant correlation with PFS could be established for high infiltration of T-follicular helper cells  and activated B cells  in the sintilimab group .In total, 118 patients , one pathway was significantly associated with improved OS only in the chemo group (glucose metabolism) and one pathway was significantly associated with improved OS in both groups  and circulating cell-free DNA  sequencing was performed at baseline for patients in the sintilimab group in order to derive TCR clonality and molecular tumor-burden index (mTBI). Our analyses showed that patients with low mTBI had a significantly longer PFS  compared with those with high mTBI. Similar trend was also observed for OS . In contrast, TCR clonality alone could not efficiently predict the clinical outcome , , and  from Fleming–Harrington test are less than 0.01, indicating the delayed effect of the significant OS benefit with sintilimab over chemo. Consequently, it is suggested that there are a delayed response and more durable benefit of immunotherapies based on similar studies and different statistical methods. The survival advantage of sintilimab as second treatment for patients with ESCC may be demonstrated via a prolonged follow-up duration.RMST is a statistical technique recently adopted widely as an alternative to median to provide an intuitive interpretation of the data. It is especially more appealing than median survival time when the data violate proportional hazard assumption when only evaluating median difference could be very misleading. The superior RMST of sintilimab over chemo was increased from 0.3 months at 9 months after randomization to 1.6 months at 18 months after randomization, further suggesting a delayed survival benefit of sintilimab. In addition, 19. Notably, for patients who were initially evaluated as PD per RECIST v1.1 in the sintilimab group, those continuing to receive sintilimab had a remarkable increase on median OS, compared with those who discontinued treatment. This finding suggested that continuous treatment with sintilimab could have a long-term survival benefit and should be further explored for patients after initial PD by RECIST v1.1.Moreover, the ORR of patients with sintilimab was twice higher than those with chemo, similar to the findings in the other four studies19. In particular, three treatment-related deaths, which were due to upper gastrointestinal bleeding, pneumonitis, and lung infection, occurred in the sintilimab group. Pneumonitis and lung infection were previously reported in PD-1 inhibitor-treated patients with esophagus cancer19. Nevertheless, upper gastrointestinal bleeding is one of the most common fatal causes for ESCC. Upper gastrointestinal bleeding was considered to be treatment-related, perhaps because this study was an open-label study.Sintilimab showed a favorable safety profile over chemo, with a substantially reduced incidence of TRAEs (54.3 vs. 90.8%) and TRAEs of grade 3 or worse (20.2 vs. 39.1%). Most toxic effects were comparable to the historical data on treatment with sintilimab in other indications and to that of treatments with other PD-1 inhibitors in esophagus cancerIn this study, comprehensive biomarker analyses were performed, including tumor PD-L1 expression, NLR, tumor transcriptome analysis and peripheral blood TCR, and cfDNA analysis.19. That is potentially attributed to limited sample availability.Even if only the HR for OS in the combined-proportion score (CPS) ≥ 10 subgroups was less than the CPS < 10 subgroups, neither PD-L1-expression level assessed by tumor-proportion score (TPS) nor CPS could predict definitely benefit from the treatment with sintilimab unlike other studies23. As is known, NLR can reflect differences in the immune status during cancer development and progression23. A meta-analysis showed that high NLR is associated with worse survival in esophageal cancer, as it is also shown in several solid tumors24. Our results revealed that low NLR (<3) at baseline or at 6 weeks post treatment with sintilimab was significantly correlated with a longer OS and PFS. The increased number of neutrophils in the peritumoral inflammatory-cell infiltrate can reduce the antitumor activity of natural-killer (NK) and activated T cells26. Moreover, many cytokines  and VEGF produced as a result of neutrophil activation may increase tumor growth27. But the mechanism is still unclear that NLR is related to survival in esophageal cancer and other solid tumors. The test of NLR is very simple and practicable, but the use of NLR in clinical practice is still underestimated.NLR is an inflammatory-response biomarker for the prognosis of ESCC in patients treated with chemoradiotherapy and immune-checkpoint inhibitors30. Furthermore, T-follicular helper cells have been shown to play important roles in the generation of TLS in the tumor microenvironment31, which might be relatively enriched for B-cell signatures and TLS in ESCC32. Thus, the potential relevance of TLS in the mode of action of cancer immunotherapy to mount an efficient antitumor response is supported by our study and warrants further investigation. However, due to tumor-specimen limitations, additional analyses to characterize TLS and their spatial distribution within the tumor microenvironment could not be performed. The correlation of several signaling pathways with clinical efficacy is more challenging to interpret. Different reports indicate a potentially context-dependent role for Wnt/β-catenin signaling in CD8 + T-cell differentiation and memory formation34. Wnt pathway also enhances the proliferation and cytolytic activity of human γδT cells35. Autocrine Wnt pathway activation induces vascular normalization and endows endothelial-cell basement membrane the capacity to boost T-cell infiltration36. Further evidence shows that, in contrast to early-stage tumors, advanced and metastatic tumors have downregulated Wnt expression. In some cases, endogenous Wnt signaling even generally counteracts tumor progression37. Also, the small sample size could contribute to these surprising findings. Such conflicting data clearly underline the need for additional analyses and validation of the data.Although general T-cell tumor infiltration did not show a predictive effect in the analysis of tumor transcriptomes, infiltration of T-follicular helper cells and activated B cells was significantly related to PFS in the sintilimab group and not in the chemo group. Especially, the predictive value of activated B lymphocytes is supported by an emerging role of B cells and tertiary lymphoid structures (TLS) in tumors, which have been recently reported in association with survival, even in tumor with low T-cell infiltration38. Whereas mTBI is derived from peripheral blood, tumor mutation burden (TMB) is generally derived from mutation assessment in tumor tissue. In contrast to TCR clonality, mTBI index was significantly associated with clinical efficacy in the treatment with sintilimab. However, the predictive value of mTBI in combination with TCR clonality might be further enhanced as patients with low mTBI and high TCR clonality exhibited the longest survival in the sintilimab group. TCR-clonality analyses revealed conflicting results in previous studies, potentially due to the dilution effect of non-tumor-specific TCRs since this analysis alone cannot distinguish between tumor vs. nontumor specificity41. However, a high clonality might be indicative of a preexisting and expanded tumor-specific T-cell response that has been blocked or exhausted. It is intriguing to speculate that such a block is mediated by the PD-1 axis and sintilimab treatment might release this block. However, reinvigoration and subsequent tumor eradication seems to mainly occur in patients with low mTBI. Detailed mechanisms for this correlation need to be examined, but apart from physical barriers in larger tumors it was previously reported that the ratio of T-cell reinvigoration to baseline tumor burden instead of the magnitude of the reinvigoration alone might be a better indicator of response43. Additionally, it has been reported that skewed ratios of infiltrating suppressive immune cells like myeloid-derived suppressor cells and T regulatory cells vs. T-effector cells, dendritic cells, and natural-killer cells correlate with tumor progression and increased tumor burden. Such cellular changes are accompanied with increasingly immune-suppressive cytokine milieus in the growing tumors48. Our data further support the notion that an effective mounting of an antitumor response is multifactorial and does not only rely on successful reinvigoration of exhausted T cells, but also on tumor burden and preexisting TCR clonality in addition to previously described predictive biomarkers like PD-L1 expression, TMB, and T-cell infiltration. It remains to be investigated if these factors are independent contributors or surrogates of each other.In contrast to the analysis of tumor tissue, the investigation of peripheral blood has practical advantages without the limitation of tumor tissue accessibility and size. We found that peripheral TCR clonality at baseline did not predict the efficacy of sintilimab. In addition, we also analyzed mTBI as an indicator of tumor burden at the molecular levelThis study had several limitations. First, the sample size of patients was relatively small. Second, the open-label design of the study might have influenced the assessment of the incidence of adverse effects. Nevertheless, this design was deemed acceptable because both dose regimens and toxicities in the two groups were disparate. Third, it was hard to evaluate the correlation between the expression of PD-L1 and the survival benefit of sintilimab due to the limited tumor samples available for the assessment of PD-L1 expression.In conclusion, the results of ORIENT-2 study favored the use of sintilimab over chemo in Chinese patients with advanced ESCC refractory to previous chemotherapies, as it suggested a prolonged survival benefit and a favorable safety profile. Both NLR <3 at 6 weeks post treatment and the combination of high TCR clonality with low mTBI may be potent biomarkers for the prediction of improved OS and PFS in patients with ESCC treated with sintilimab.This was a multicenter, randomized, parallel, open-label phase-2 study conducted at 30 sites in China Table . PatientPatients were randomly (1:1) assigned to receive either sintilimab or the investigator’s choice of chemo (paclitaxel or irinotecan), using an interactive web-response system with a block size of a mixture of 2, 4, and 6, and with the stratification factor of the ECOG PS score (0 vs. 1). Neither investigators nor patients were blinded to treatment allocation.2 intravenous paclitaxel once every 3 weeks, or 180 mg/m2 intravenous irinotecan once every 2 weeks), until disease progression, death, unacceptable toxicity, or withdrawal of informed consent.After randomization, patients were treated with 200 mg of intravenous sintilimab, once every 3 weeks, or with the investigator’s choice of chemo . Adverse events (AEs) and serious adverse events (SAEs) were monitored throughout the treatment period and for 90 days after the last dose, based on the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE v4.03).The expression of PD-L1 was assessed via immunohistochemistry (Dako PD-L1 IHC 22C3 pharmDx) using the Autostainer Link 48 . PD-L1 protein staining was determined using the tumor-proportion score (TPS) or the CPS.49.The NLR inflammatory biomarker, which was calculated as neutrophil count/lymphocyte count, was obtained from hematology panels at baseline and 6 weeks post treatment. High and low NLR groups were classified using the cutoff of NLR = 350. Clean reads were obtained through trimming clean reads using Trimmomatic (version 0.36)51. STAR aligner (version 2.7.0a) was used to map clean reads to human reference genome (hg38)52. HTSeq (version 0.11.4) was used for counting reads mapped to genes53. The gene sets defining 28 immune-cell populations were extracted from the previous research38. The gene sets of 45 signaling pathways were downloaded from the tumor signaling 360 panel of NanoString . The tumor samples of biomarker-evaluable population (BEP) with qualified RNA-seq data were used to conduct downstream analysis. To screen for survival-related genes, based on transcript-per-million (TPM)-normalized gene expression level of each protein-coding gene, the BEP was iteratively split into high or low expression groups (Shigh_low) by median value of the whole cohort of the respective gene. Then, Cox regression model by the formula Surv  ~Shigh_low in each of the treatment arm was run using the R survival package (version 2.44-1.1), to derive the respective hazard ratio and raw P-value54. All genes were ranked by raw P-value, whereas genes with P < 0.05 were considered as potentially survival-related genes. Gene-set signature scores were calculated using the GSVA algorithm (version 1.32.0) based on the gene expression levels of TPM55. High and low signature scores were split according to the median value of the whole cohort. Then the same screening procedure as described above was conducted.Quality control of RNAseq raw data was performed using FastQC (version 0.11.8)38. Sequencing data of ctDNA were mapped to human reference genome (hg19) by using BWA (version 0.7.17)56. GATK (version 4.1.4.1) was used to call variants of each sample57. PyClone (version 0.13.1) was used to analyze the clonal-population structures based on baseline-mutation profile58. Sequencing reads of TCRβ CDR3 region were merged to obtain contigs using Pear (version 0.9.11)59, and then contig sequences were aligned to reference TRB V/(D)/J gene sequences (http://www.imgt.org) using MiXCR (version 3.0.4) to determine the TRB V/(D)/J gene segment in each contig60. High and low TCR clonality or mTBI of baseline samples were split according to the median value of the whole cohort. Then, Cox regression model by the formula Surv ~ Shigh_low in each of treatment arm was run using the R survival package (version 2.44-1.1)54, to derive the respective hazard ratio (HR) and raw P-value (P). The ‘time’ represents PFS or OS, ‘event’ for progression or death event, and ‘Shigh_low’ for split groups by median value. Survival curves were plotted using the R survminer package (version 0.4.6)61.Peripheral baseline T-cell receptor (TCR) CDR3 regions and cell-free DNA were sequenced to calculate TCR clonality and molecular tumor-burden index (mTBI) in each sample. The latter is a reflection of the percentage of circulating-tumor DNA (ctDNA) detected in whole cell-free DNA (cfDNA) and is considered to reflect the tumor burden at the molecular level, which is defined as the mean allele fraction of mutations in the trunk cluster as published earlierThe primary endpoint was OS, defined as the time from randomization until death. Surviving patients were censored using the data at the last follow-up. Secondary endpoints were PFS, which was the time from randomization to initial disease progression evaluated by the investigator per RECIST v1.1 or death; ORR, defined as the percentage of patients achieving complete response (CR) or partial response (PR); DCR, which was the percentage of patients with the best overall response of CR, PR, or stable disease (SD); time to response (TTR); and duration of response (DOR). As safety endpoint was considered the incidence of all AEs across the study. Exploratory endpoints were the association between efficacy (OS and PFS) and biomarkers, including PD-L1 expression, mRNA expression, and ctDNA results. The association between efficacy  and NLR was post hoc analysis. Health-related quality of life (HRQoL) was assessed by the European Organisation for Research and Treatment of Cancer Quality-of-Life Questionnaire-Core 30 (EORTC QLQ-C30), and the five-level version of European Quality of Life-5 Dimensions (EQ-5D-5L) visual analogue scale (VAS), at the first dose, as well as at every radiological evaluation time and at the time of the first safety follow-up.α = 0.2, two-sided) under a statistical power of 80%. Assuming a 21% dropout rate, 180 patients were required.The required death-event number was 142, with an expected HR for OS of 0.7 (All efficacy endpoints were assessed in the intention-to-treat (ITT) population that included all randomly assigned patients. The safety profile was assessed in the safety set (SS), which included patients receiving at least one dose of the study drug.The Kaplan–Meier method was utilized to evaluate the time-to-event endpoints , and the differences in the survival curves between the two groups were analyzed using the stratified log-rank test by ECOG PS score. A stratified Cox proportional hazard regression model was used to estimate the HR and corresponding 95% confidence interval (CI) of OS and PFS between treatment groups, using the ECOG PS score (0 vs. 1) as a stratification factor. The RMST was evaluated to compare the differences in OS between groups at 9, 12, 15, and 18 months post treatment. The binomial distribution method was used to evaluate the 95% CI of ORR and DCR, and differences were compared using the Cochran Mantel–Haenszel test. Patient-reported health-related quality-of-life outcomes were analyzed for patients who had a baseline and at least one post-baseline assessment of EORTC QLQ-C30  and EQ-5D-5L (VAS and index).(ρ–γ), which accounts for nonproportional hazards . Considering the potential pseudoprogression for PD-1 inhibitors, a post hoc analysis was performed to compare the difference in OS between patients with and without continuous treatment of sintilimab after initial PD per RECIST v1.1 in the sintilimab group.To evaluate any factors correlated with the crossing of the survival curves in OS, different treatments were assessed using a weighted log-rank test from the FH Gα = 0.05 (two-sided) and the significance threshold was P < 0.05. For biomarker identification using bioinformatics, survival analysis was performed using the survival package43 in R, by which both the P-value and hazard ratio between two groups were calculated using the coxph function. Survival curves were plotted using the survminer package44.Statistical analyses for the clinical part were performed using the SAS software (version 9.4) . The significance level for all endpoints was Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary